GPR50 regulates neuronal development as a mitophagy receptor

Neurons rely heavily on high mitochondrial metabolism to provide sufficient energy for proper development. However, it remains unclear how neurons maintain high oxidative phosphorylation (OXPHOS) during development. Mitophagy plays a pivotal role in maintaining mitochondrial quality and quantity. We herein describe that G protein-coupled receptor 50 (GPR50) is a novel mitophagy receptor, which harbors the LC3-interacting region (LIR) and is required in mitophagy under stress conditions. Although it does not localize in mitochondria under normal culturing conditions, GPR50 is recruited to the depolarized mitochondrial membrane upon mitophagy stress, which marks the mitochondrial portion and recruits the assembling autophagosomes, eventually facilitating the mitochondrial fragments to be engulfed by the autophagosomes. Mutations Δ502-505 and T532A attenuate GPR50-mediated mitophagy by disrupting the binding of GPR50 to LC3 and the mitochondrial recruitment of GPR50. Deficiency of GPR50 causes the accumulation of damaged mitochondria and disrupts OXPHOS, resulting in insufficient ATP production and excessive ROS generation, eventually impairing neuronal development. GPR50-deficient mice exhibit impaired social recognition, which is rescued by prenatal treatment with mitoQ, a mitochondrially antioxidant. The present study identifies GPR50 as a novel mitophagy receptor that is required to maintain mitochondrial OXPHOS in developing neurons.


INTRODUCTION
Mitochondria, as the powerhouse of eukaryotic cells, supply neurons with energy by generating metabolites via the tricarboxylic acid (TCA) cycle and ATP through OXPHOS.Neurons, characterized by their high energy demands, heavily rely on ATP provided by mitochondria [1].Mitochondrial metabolism controls the species-specific tempo of cortical neurons.The prolonged human neuronal development correlates with lower mitochondrial metabolic activity, particularly that of OXPHOS.Enhancing mitochondrial oxidative metabolism accelerates human neuronal development, further emphasizing the essential role of OXPHOS in neuronal development [2].Neurons cannot generate ATP through glycolysis, making them more susceptible to mitochondrial dysfunction [3].Mitochondrial dysfunction results in insufficient ATP supply, oxidative stress, and impaired signaling pathways, which have been linked to neurodevelopmental disorders such as autism spectrum disorders (ASD).The latter is clinically manifested with impaired social capability and repetitive and stereotyped behaviors [4,5].Mitochondrial dysfunction and elevated ROS levels are even considered hallmarks of ASD [6][7][8][9].However, how neurons maintain high mitochondrial OXPHOS to provide sufficient energy for proper development remains unclear.
Mitophagy, a subtype of selective autophagy, is pivotal in maintaining mitochondrial quality and quantity.Mitophagy relies on a population of factors called mitophagy receptors to recruit the assembling autophagosomes (phagophores) adjacent to the damaged mitochondria.With the fusion of autophagosomes to lysosomes, the engulfed mitochondria are degraded.Mitophagy receptors usually contain a conserved LC3 or GABARAP interaction region (LIR or GIM, respectively), through which mitophagy receptors bind to LC3 and recruit the assembling autophagosomes.The LIR/GIM motif is characterized by conserved "W/F/Y-xx-L/I/V" motifs surrounded by acidic amino acids in the cytoplasmic domain [3].Intriguingly, mitophagy is broadly activated in metabolically enhanced neurons.OXPHOS stimulation depolarizes the mitochondrial membrane and enhances mitophagy.These data indicate that to sustain high energetic activity, neurons must increase mitochondrial turnover and, hence, facilitate mitochondrial maintenance [3].However, how mitophagy is coordinated with mitochondrial OXPHOS to control neuronal development remains unknown.
G protein-coupled receptor 50 (GPR50), an X-chromone-linked orphan GPCR, shares approximately 45% of its amino acid sequence homology with the melatonin receptors MT1 and MT2.However, GPR50 does not bind to melatonin or any other ligand, therefore remaining an orphan receptor [10].Two GPR50 variations, Δ502-505 and T532A, have been detected in patients with ASD [11,12], bipolar disorder, and major depression [13][14][15].However, limited information is known about the role of GPR50 in the brain.Knocking down GPR50 in vitro decreases the proliferation and differentiation of neural progenitor cells and neurite outgrowth [16,17], suggesting that GPR50 plays a role in maintaining neuronal development and function.We herein describe GPR50 as a novel mitophagy receptor containing an LIR and binding to LC3.GPR50 is recruited to the depolarized mitochondrial membrane upon mitophagy stress, where it marks the mitochondrial portion for degradation and recruits the assembling autophagosomes, facilitating the mitochondrial portion to be engulfed by the autophagosomes.We further show that disease-related mutations at Δ502-505 and T532A impair GPR50mediated mitophagy by attenuating the binding of GPR50 to LC3 and the mitochondrial recruitment of GPR50.GPR50 deficiency causes accumulation of the damaged mitochondria, impairing mitochondrial OXPHOS, resulting in insufficient ATP production and excessive ROS accumulation, eventually leading to defective neuronal development.GPR50-deficient mice exhibit impaired social recognition, which is rescued by prenatal treatment with mitoQ, a mitochondrially antioxidant.Thus, GPR50, as a mitophagy receptor, is required for neuronal development via maintaining mitochondrial OXPHOS.

GPR50 is transiently recruited to the mitochondrial region upon mitochondrial membrane depolarization
We failed to detect the distribution of GPR50 in the mitochondria in HEK293 cells under standard culturing conditions using immunofluorescence staining (Supplementary Fig. 1A, B).To mimic the mitophagy stress during OXPHOS stimulation [3], we treated HEK293 cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Fig. 1).This mitochondrial uncoupler induces mitochondrial depolarization and mitophagy in a time-dependent manner [18,19].TOMM20 levels (a mitochondrial outer membrane protein) and LC3-II in the whole cell lysates (Fig. 1A, B, D; Supplementary Fig. 4) were decreased and increased respectively from 2 h of CCCP treatment.The levels of LC3-II in the mitochondrial fraction, which marks the autophagosomes/phagophores recruited by mitochondria, were increased from 2 h to reach a peak of 6 h of CCCP treatment (Fig. 1A, D).We were surprised to observe a transiently and dynamic mitochondrial distribution of GPR50 upon CCCP treatment, which displays a synchronous pattern with CCCP-induced mitophagy.The endogenous GPR50 in the mitochondrial fraction accumulated from 2 h to peak at 6 hours and declined from 12 h of CCCP treatment (Fig. 1A, C).Coimmunostaining for FLAG and TOMM20 in HeLa cells showed similar results (Fig. 1E, F).These results indicate that GPR50 is transiently recruited to the mitochondrion upon the depolarization of the mitochondrial membrane.The high synchronicity between the mitochondrial recruitment of GPR50 and autophagosomes/phagophores suggests a role of GPR50 in mitophagy.
GPR50 binds to LC3 via the LIR motif, which is attenuated by ASD mutations Autophagic receptors necessitate dimerization or oligomerization for optimal functionality in selective autophagy.Dimerization or oligomerization increases the concentration of autophagy receptors in the local membrane, enhancing the binding affinity of these factors to LC3 or membranes, thus facilitating their function in membrane curvature and recruiting phagophores [20].Thus, to identify the molecular mechanism underlying GPR50-mediated mitochondrial function, we used biomolecular complementation affinity purification (BiCAP) to screen the factors potentially interacting with GPR50 homodimers.The carboxyl terminus of GPR50 was linked to the V1 or V2 segment of the fluorescent Venus protein.The GPR50-V1 and GPR50-V2 homodimers were immunoprecipitated and subjected to liquid chromatography and mass spectrometry (LC-MS) analysis (Fig. 2A).The interaction partners of GPR50 homodimers were subjected to Gene ontology (Go) analysis.The cellular components from Go terms showed an extensive location in the mitochondrial membrane, matrix, ribosomes, and mitochondrial respiratory complex, which further confirms a distribution of GPR50 in mitochondria.The biological processes from Go terms include pathways related to OXPHOS, autophagy, mitophagy, and responses to oxidative stress (Fig. 2C).We observed an extensive interaction of GPR50 with various mitophagy regulators including mitophagy receptors (FKBP8, PHB2, CERS1) and autophagy receptor (SQSTM1), kinases that modulate phosphorylation (PGAM5, PARK7, CSNK2, MARK2, HK2) and ubiquitination (AMFR, HUWE1) of mitophagy receptors, mitochondrial shaping proteins (FIS1 and OPA1).Of note, LC3 was also identified as one of the binding partners of GPR50 (Fig. 2B).Because GPR50 forms a protein complex with various mitophagy regulators, we were curious whether GPR50 directly binds to LC3.Autophagy receptors are characterized by a conserved "W/F/Y-x-x-L/I/V" motif surrounded by acidic amino acids [21].Analysis of the sequence of GPR50 protein by iLIR [22] indicates that GPR50 contains one conserved "W/F/Y-x-x-L/I/V" motif (LIR, Y 305 WTI 308 ) in its cytoplasmic domains (Fig. 2D, E).To examine whether a direct binding exists between Y 305 WTI 308 and LC3, we performed ELISA by using recombinant LC3 protein coated on the dish plate to bait biotin-labeled synthetic peptides comprising of LIR, e.g., a.a.290-312.The results showed that LIR peptides bound to recombinant LC3 in a dose-dependent manner, which was abolished by mutating Y 305 WTI 308 into quadruple alanine (Fig. 2D, F).The PLA-ligation assay in HeLa cells transfected with WT-GPR50-FLAG further showed that GPR50 interacts with endogenous LC3 (Fig. 2G, H).Co-IP analysis with the lysates from the cells cotransfected with GPR50-FLAG and LC3-GFP observed that both LC3-I and its lipidated form, LC3-II [23], were immunoprecipitated by GPR50-FLAG (Fig. 2I, J; Supplementary Fig. 4).The endogenous interaction between GPR50 and LC3 was further confirmed by the Co-IP assay with lysates from mouse brains (Fig. 2K; Supplementary Fig. 4).Both Co-IP and the duolink assay showed that mutation on the Y 305 WTI 308 motif attenuated the binding of GPR50 to LC3, indicating that Y 305 WTI 308 is one of the LIR motifs in GPR50 (Fig. 2F-J).

Mitochondrial recruitment of GPR50 does not depend on its binding to LC3
We further examined whether the mitochondrial recruitment of GPR50 relies on its binding to LC3.HeLa cells were transfected with WT GPR50, GPR50 T532A , GPR50 Δ502-505 , or mLIR plasmids, which were fused a FLAG tag in their carboxyl terminus and treated with CCCP.WB analysis with an anti-FLAG antibody showed that compared to WT GPR50, the levels of GPR50 T532A and GPR50 Δ502-505 , but not those of GPR50 mLIR , were decreased in the mitochondrial fraction in CCCP-treated HeLa cells, while their levels in the whole cell lysates remained comparable to GPR50 WT (Fig. 3A, B; Supplementary Fig. 4).Coimmunostaining analysis revealed that in comparison to WT GPR50, GPR50 T532A and GPR50 Δ502-505 , but not those of GPR50 mLIR , displayed decreased colocalization with TOMM20 in CCCP-treated HeLa cells (Fig. 3C, D).Therefore, these results indicate the mitochondrial recruitment of GPR50 does not rely on its binding to LC3.In contrast, ASD mutations impair the mitochondrial recruitment of GPR50 in a mechanism independent of binding to LC3.

GPR50 is required in CCCP-induced mitophagy in an LC3binding dependent manner
Because the mitochondrial recruitment of GPR50 is highly synchronous to that of autophagosomes and GPR50 contains LIR, we wondered whether GPR50 is a novel mitophagy receptor.HeLa cells cotransfected with either GPR50 siRNA or a scrambled siRNA (NC) (Supplementary Fig. 2A; Supplementary Fig. 5), GFP-LC3, and mitoDsRed were treated with CCCP to induce mitophagy (Fig. 4A).GPR50 siRNA abolished the CCCP-induced accumulation of mitophagosome (LC3 + mitoDsRed + puncta) (Fig. 4A, B), while it attenuated the CCCP-induced mitochondrial fragmentation (Fig. 4A, C), indicating that GPR50 is required in the CCCPinduced mitophagy.Notably, the knocking-down of GPR50 failed to affect the mitophagy in control cells treated with DMSO (Fig. 4A-C), suggesting that GPR50 mediates mitophagy upon mitochondrial depolarization rather than consecutively.
We also observed that the overexpression of GPR50 is sufficient to induce mitophagy in HeLa cells by using a similar experimental approach (Supplementary Fig. 2B-D).Overexpression of GPR50 reduced the protein levels of mitochondrial proteins, including TOMM20 (a mitochondrial preprotein translocase anchored in the outer membrane), TIM23 (a mitochondrial preprotein translocase anchored in the inner membrane), and MTCO2 (one of the components of the cytochrome c oxidase) in HeLa cells, which was prevented by the treatment with bafilomycin A1 (Fig. 4D-H; Supplementary Fig. 5; Supplementary Fig. 2E-J; Supplementary Fig. 5), an inhibitor of the lysosomal proton pump to inhibit lysosomal acidification and to block the fusion of autophagosomes to lysosomes [24].In contrast, overexpression of GPR50 failed to alter the levels of Mtco2 mRNA (Supplementary Fig. 2H), further confirming a posttranscriptional mechanism involved.
These results indicate that GPR50, a novel mitophagy receptor, is recruited to the depolarized mitochondria, which marks the damaged mitochondria and recruits the assembling autophagosomes via binding to LC3.Via this way, GPR50 and the damaged mitochondria are engulfed by autophagosomes for further degradation.Mutations of GPR50 at either T532A or Δ502-505 attenuate this process.

Deficiency of GPR50 impairs mitochondrial metabolism and increases ROS levels
Immunofluorescence staining on the mouse cortical and hippocampal sections and the primary cultured neurons revealed that neurons predominantly express GPR50.Few GPR50 was detected in astrocytes (Supplementary Fig. 1C-F).The specificity of the GPR50 antibody was validated by the absence of GPR50 immunoreactivity in Gpr50 −/y brains and HEK-GPR50 KO cells (Supplementary Fig. 1G, H; Supplementary Fig. 5).As GPR50 plays an essential role in mitophagy, we further analyzed whether a GPR50 deficiency would cause mitochondrial dysfunction.Both GPR50-deficient HEK293T cells (Fig. 5A, C) and mouse primary cortical neurons (Fig. 5B, D) exhibited a striking decrease in the fluorescent density of TMRE, an established marker for mitochondrial membrane potential [27], indicating GPR50 deficiency results in the accumulation of depolarized mitochondria in these cells.These phenomena were rescued by transfection of WT GPR50 plasmid but not by transfection of GPR50 mLIR or GPR50 ASD mutants (GPR50 T532A , GPR50 Δ502-505 ) (Fig. 5A-D), indicating GPR50 maintains mitochondrial quality in a mechanism dependent on its binding to LC3.TEM analysis unveiled that the mitochondria in Gpr50 −/y neurons exhibited notable vacuolization and swelling within the cristae (Fig. 5E, F).The mitochondrial protein TOMM20 was accumulated in the brains of adult Gpr50 −/y mice (Fig. 5G, H).These results indicate that GPR50 deficiency results in the accumulation of the damaged mitochondria.

Deficiency of GPR50 results in defective social behaviors in mice
Adult Gpr50 −/y mice exhibited normal fertility but a smaller body size than Gpr50 +/y mice (Supplementary Fig. 2M).Both Gpr50 −/y and Gpr50 +/y mice can survive into adulthood.Considering the genetic link of GPR50 to ASD, depression, and bipolar disorders [11,12], all of which display impaired social interaction, we examined whether a deficiency of GPR50 is linked to defective social behaviors by subjecting the mice to a three-chamber test (Supplementary Fig. 3A).In the first phase of the three-chamber test, both adult Gpr50 +/y and Gpr50 −/y mice exhibited a preference for the stranger mice as evidenced by their spending a more extended time staying in the chamber containing the stranger mice (S) versus the one containing the empty cage (object) (Fig. 7B; Supplementary Fig. 3B) and in interacting with the stranger mice (S) versus with the empty cage (object) (Fig. 7C; Supplementary Fig. 3C), suggesting normal sociability of Gpr50 −/y mouse.In the second phase of three-chamber tests, the test mice were subjected to the chambers containing the stranger mice (S) and the familiar mice (F) on either side, respectively.In contrast to the adult Gpr50 +/y mice, which still exhibited a preference for the stranger mice (S) versus the familiar mice (F), Gpr50 −/y mice lost such preference as reflected by they spent comparable time in staying in the chamber containing the stranger mice (S) versus in the one containing the familiar mice (F) (Fig. 7D; Supplementary Fig. 3D), and in interacting with the stranger mice (S) versus the familiar mice (F) (Fig. 7E; Supplementary Fig. 3E).Gpr50 −/y and Gpr50 +/y mice displayed similar olfactory function in the buried food test, excluding the possibility that the social discrepancy was ascribed to the olfactory dysfunction of Gpr50 −/y mice (Supplementary Fig. 3F).Thus, a deficiency of GPR50 results in impaired social recognition and memory in mice.
The administration of MitoQ prenatally rescues autism-like behavior in GPR50-deficient mice ROS is predominantly produced by mitochondria and is a byproduct of OXPHOS [30].ROS acts as a signaling factor at low levels and is required in neuronal development, such as neuronal polarity, growth cone pathfinding, neurite outgrowth, and synaptic plasticity [31].In contrast, excessive ROS accumulation impairs neuronal development and synaptic plasticity, leading to neurodegeneration [31].Intriguingly, excessive ROS forms a vicious cycle with defective mitophagy.Excessive ROS activates mitophagy to accelerate mitochondrial turnover.Defective mitophagy, in turn, results in excessive ROS production.We wondered whether the elimination of the overproduction of ROS would terminate the vicious cycle between defective mitophagy and ROS and bring beneficial effects on GPR50-deficient mice.MitoQ, a mitochondrially targeted antioxidant, effectively blocks ROS and prevents mitochondrial oxidative damage [29].Thus, we administered orally Gpr50 −/y mice with mitoQ starting at E14, an active developmental stage for neurons, by feeding their pregnant mother mice with mitoQ until they were weaned.Then, Gpr50 −/y pubs were administered orally with MitoQ until they were subjected to behavioral tests (Fig. 7A).The results showed that administration of mitoQ prenatally rescued the impaired social recognition of Gpr50 −/y mouse as evidenced by that mitoQtreated Gpr50 −/y mice regained the preference for the stranger mouse versus the familiar mouse in the three-chamber social interaction test (Fig. 7B-E).Thus, the impaired social recognition caused by the defective GPR50-mediated mitophagy is prevented by eliminating excessive ROS at the early developing stage.

DISCUSSION
We herein identify that GPR50 is a novel mitophagy receptor, which is required in mitophagy stress-induced mitophagy.GPR50 is recruited to the mitochondria upon mitophagy stress, where it marks the damaged mitochondria and initiates the recruitment of the assembling autophagosomes (phagophores), facilitating the damaged mitochondria to be engulfed by autophagosomes.In this way, GPR50 maintains mitochondrial OXPHOS in the developing neurons.Deficiency of GPR50 impairs mitochondrial OXPHOS, which results in an insufficient production of ATP and an overproduction of ROS, eventually leading to delayed neuronal development and defective social recognition.The prenatal administration of mitoQ, a mitochondrial antioxidant, rescues the defective social behaviors of GPR50-deficient mice.Both Δ502-505 and T532A, two GPR50 variations genetically associated with ASD, depression, and bipolar disorders, attenuate the binding of GPR50 to LC3 and the mitochondrial recruitment of GPR50, impairing GPR50-mediated mitophagy and neuronal development (Fig. 7F).This study provides a novel mechanism underlying how developing neurons utilize mitophagy to maintain mitochondrial OXPHOS.
We have observed that the mitochondrial recruitment of GPR50 does not rely on its binding to LC3.Intriguingly, both Δ502-505 and T532A GPR50 variations, which attenuate the binding of GPR50 to LC3, impair the mitophagy stress-induced mitochondrial recruitment of GPR50, suggesting that other mechanisms are involved.The damaged mitochondria initiate a series of signal cascades on their surface, such as PINK1-mediated phosphorylation and PARKIN-mediated ubiquitination, which leads to the recruitment of autophagy machinery to the mitochondria with the aid of mitophagy receptors [3].In this context, among the binding partners of GPR50 identified by BiCAP, OPA, VCP, and PARK7 are also translocated to the depolarized mitochondria.RHOT1 and RHOT2 are required for mitochondrial damage-induced PARKIN translocation to mitochondria [51].We surmise a similar mechanism underlying the mitochondrial recruitment of GPR50, which requires further investigation.

Mice
Gpr50 −/− mice were generated by replacing the second exon with the Lacz-neo cassette and maintained on a C57BL/6 N background by crossing heterozygous transgenic mice with C57BL/6 N breeders.Gpr50 −/y mice were obtained by hybridization between Gpr50 −/− mice and Gpr50 +/y mice.For PCR genotyping, the following primers were used (F1: 5'-ATCCGGGGGTACCGCGTCGAG-3'; R1: 5'-TACCTCCACCTCCTCCAGCAT-3'; F2: 5'-CAGAGTCACCTGGGACTTGCT-3'; R2: 5'-CACAACGGGTTCTTCTGTTAGTCC-3'; F3: 5'-CAGAGTCACCTGGGACTTGCT-3; R3: 5'-GTAGCAGTAACGGTT-GATGGCAATG-3').A product size of 390 bp was obtained for the WT mouse.The product sizes of 353 bp and 357 bp were identified for GPR50 knockout mice together.All mice were group-housed with 3-5 same-sex cage mates in standard mouse cages in a pathogen-free barrier facility on a 12-h light-dark cycle with lights on at 07:00 and a controlled temperature range of 22-25 °C.Food and water were provided ad lobitum.Behavioral tests were performed during the light phase.All experimental procedures were preapproved by the Ethics Committee of Soochow University and conformed to the Institutional Animal Care and Use Committee guidelines of Soochow University (reference number: 202211A0519).

Generation of GPR50 CRISPR-Cas9 KO cell lines
For the generation of GPR50 KO HEK293T cells, single guide RNAs (sgRNAs) were designed using the online CRISPR design tool (Red CottonTM, Guangzhou, China, https://en.rc-crispr.com/).The exon region of GPR50 was selected to be targeted by CRISPR/Cas9 genome editing.A ranked list of sgRNAs was generated with specificity and efficiency scores.The pairs of oligos for two targeting sites were annealed and ligated to the YKO-RP006 vector (Ubigene Bioscience Co. Ltd., Guangzhou, China).The YKO-RP006-hRABL6 [gRNA] plasmids containing each target sgRNA sequence were transfected into cells with Lipofectamine 3000 (Thermo Fisher Scientific).24-48 h after the transfection, puromycin was added to screen the cells.After antibiotic selection, cells were diluted using the limited dilution method and inoculated into a 96-well plate.Single GPR50 KO clones were performed after being cultured for 2-4 weeks and validated by PCR, western blotting, and Sanger sequencing.

Drug treatment
The gpr50 −/y and gpr50 -/+ mice were allowed to mate at 5 pm.The vaginal plugs were checked at 8 am the next day.The mice with vaginal plugs were supposed to be in pregnancy for 0.5 days.The gpr50 -/+ mice that were pregnant for 14 days were administrated with mitoQ (New Zealand, 70443110246) orally at 5 mg/kg/d, which were mixed into the food chaw.The pubs were genotyped after birth, and gpr50 −/y pups were fed food containing mitoQ until they were subjected to behavioral tests at 2 months old.Gpr50 +/y and gpr50 −/y mice, fed food with normal saline, served as controls.

Analysis of colocalization of LC3 and MitoDsRed and fragmented mitochondria
The quantification was performed as described previously [19].HeLa cells co-transfected with LC3-GFP and MitoDsRed plasmid were imaged with a confocal microscope.The colocalization ratio of LC3-GFP and MitoDsRed were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD) as described.After correcting the background, Pearson's correlation coefficient was calculated as the colocalization ratio.The mitochondrial fluorescent signals display as network or baculiform shape in normal conditions.We used Image J to measure normal and fragmented mitochondria.Considering the network structure formed through mitochondrial fusion and fission, we assessed a minimum of 15 mitochondria per cell and computed their average length.Mitochondria with an average length equal to or less than 5 μm were categorized as undergoing fragmentation, whereas those exceeding 5 μm were deemed normal.The percentage of cells with fragmented mitochondria was counted [66,67].

Proximity ligation assay (PLA)
Cells were transfected with GPR50 WT, GPR50 LIR mutant, and ASD mutant plasmids for 30 h.Cells were then fixed with 4% paraformaldehyde for 20 min at RT. Cells were permeabilized in PBS containing 0.3% triton for 5 min, then washed with PBS twice.After being blocked in Duolink block solution for 2 h at RT, cells were incubated with anti-LC3 (Novus, NB100-2220) and mouse anti-FLAG (Sigma-Aldrich, F3165) antibodies which were diluted in Duolink antibody dilution buffer overnight at 4 °C.The following morning, cells were washed for 10 min in washing buffer A, followed by adding the appropriate Duolink secondary antibodies (Sigma-Aldrich, DUO92004) diluted and mixed according to the manufacturer's instruction.Cells were incubated for 30 min at 37 °C, after which cells were washed with washing buffer A twice, each 5 min.Ligation and amplification steps of the PLA were performed using the Duolink in situ Green Starter kit (Sigma-Aldrich, DUO92014) according to the manufacturer's instructions.Cells were then mounted in a Prolong Gold mounting medium with DAPI (Invitrogen, P36941).Images were acquired on a Zeiss LSM900 Confocal microscope.PLA spots were counted using Image J software (NIH, Bethesda, MA, USA).

Quantification of dendritic complexity
Primary neurons were isolated from the hippocampus and cortex of newborn mouse pubs and cultured in neurobasal-A (Gibco, 10888022) medium supplemented with 2% B27 (Gibco, 17504044), 1% Glumax (Gibco, 35050061) and 1% Penicillin-Streptomycin (P/S) at 37 °C.The cultured neurons were transfected with GPR50-FLAG and immunostained for MAP2.The images were captured by an LSM780 confocal microscope (Zeiss, Jena).Sholl analysis was performed using the Image J software under the following parameters (start radius: 2 μm, end radius: 30 μm, radius step: 5 μm).The numbers of intersections at radial intervals of 2 μm starting from the central point of the soma were identified and averaged to create the mean sholl curve.

HPLC-tandem MS (MS/MS) analysis of peptides
The peptide mixture was analyzed by a home-made 30 cm-long pulled-tip analytical column (75 μm ID packed with ReproSil-Pur C18-AQ 1.9 μm resin, Dr. Maisch GmbH), the column was then placed in-line with an Easy-nLC 1200 nano HPLC (Thermo Scientific) for mass spectrometry analysis.The analytical column temperature was set at 55 °C during the experiments.The mobile phase and elution gradient used for peptide separation were as follows: 0.1% formic acid in water as buffer A and 0.1% formic acid in 80% acetonitrile as buffer B, 0-1 min, 5%-10% B; 1-96 min, 10-40% B; 96-104 min, 40%-60% B, 104-105 min, 60%-100% B, 105-120 min, 100% B. The flow rate was set as 300 nL/min.Data-dependent MS/MS analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific).Peptides eluted from the LC column were directly electrosprayed into the mass spectrometer with the application of a distal 2.5-kV spray voltage.A cycle of one full-scan MS spectrum (m/z 300-1800) was acquired followed by top 20 MS/MS events, sequentially generated on the first to the twentith most intense ions selected from the full MS spectrum at a 30% normalized collision energy.Full scan resolution was set to 70,000 with automated gain control (AGC) target of 3e6.MS/MS scan resolution was set to 17,500 with isolation window of 1.8 m/z and AGC target of 1e5.The number of microscans was one for both MS and MS/MS scans and the maximum ion injection time was 50 and 100 ms, respectively.The dynamic exclusion settings used were as follows: charge exclusion, 1 and >8; exclude isotopes, on; and exclusion duration, 30 seconds.MS scan functions and LC solvent gradients were controlled by the Xcalibur data system (Thermo Scientific).
performed as follows: baseline, 3 cycles; injection of Rotenone/Antimycin A (final concentration: 0.5 μM), 3 cycles; injection of (final concentration: 50 mM), 5 cycles.Each cycle is composed of mix 3 min, wait 2 min, and measure 3 min.After completion of the measurements, the amount of protein within the plate was determined by BCA.Data are presented as pH changes per min in the medium using the Seahorse Wave 2.6.0 version (Agilent), normalized to total protein content.Glycolic Proton Efflux Rate (glycoPER), referring to the proton efflux rate brought by the production of lactic acid during glycolysis, was calculated as the differences between the total PER and the mitochondrial PER.Mitochondrial PER refers to the proton efflux rate from respiratory sources (contribution of mitochondrial/ TCA cycle-derived CO 2 to extracellular acidification).Mitochondrial PER was calculated by measuring OCR before and after adding Rot/AA using the Buffer Factor (BF, 2.8) and CO 2 contribution factor (CCF).

Enrichment of mitochondria
HEK293T cells were treated with 50 μM CCCP (Yeasen, China, 40333ES60) for 2, 6, and 12 h and collected.Mitochondria were isolated using the Cell Mitochondria Separation Kit (Beyotime Co., Nantong, China, C3601) as instructed by the manufacturer.Briefly, cells were collected and homogenized on ice in mitochondrial separation reagent A containing 1 mM PMSF (Beyotime Co., Nantong, China, ST506).Cell homogenates were centrifuged at 600 g at 4 °C for 10 min, and then the supernatant was collected and centrifuged at 11,000 g at 4 °C for 10 min.The precipitate was collected and resuspended in mitochondrial buffer A. The supernatant was collected as the cytoplasmic fraction depleted mitochondria.

High-resolution respirometry
After washing with pre-cooled PBS, the region of the cortex and hippocampus were dissected and put into a tube containing MIR05 respiratory buffer (20 mM HEPES, 110 mM sucrose,10 mM KH 2 PO4, 20 mM taurine, 60 mM lactobionic acid, 3 mM MgCl 2 , 0.5 mM EGTA [pH 7.1], 1 mg/ml fatty acid-free BSA, catalase 280 U/ml).The tissues were homogenized on ice, and the mitochondrial oxygen consumption rate (OCR) was measured via O 2 K (Oroboros instruments).Briefly, the tissue homogenates were placed in the respiration chamber in MIR05.After the baseline recording, the substrate pyruvate (5 mM), glutamate (10 mM), and malate (2 mM) as the substrates of complex I.When the respiration was stabilized, the leak value of complex I was obtained.The maximum oxidative phosphorylation value of complex I was obtained by adding ADP (2.5 mM).Cytochrome C (10 μM) was added to verify the integrity of the mitochondrial membrane.Succinate (10 mM) was added to obtain complexes I and II's maximum oxidative phosphorylation value.The maximum electron transport capacity was obtained by adding uncoupling agent CCCP (1.5 μM).The maximum electron transport capacity of complex II was obtained by adding rotenone (complex I inhibitor) (0.5 μM).Antimycin A (inhibitor of complex III) (2.5 μM) was added to obtain ROX non-mitochondrial respiration.Add Ascorbate (2 mM) and TMPD (0.5 mM) to detect the respiratory function of complex IV.All measurements were performed at 37 °C.

Detection of Reactive oxygen (ROS)
Cells transfected with GPR50 WT or GPR50 mutant plasmids were treated for 50 μM CCCP for 2 h.Following the manufacturer's instructions, the ROS test was determined using a ROS assay kit (Beyotime, S0033S). 10 5 cells were incubated with CellROX (5 μM) in a cell incubator for 30 mins at 37 °C and washed three times with PBS.Cells were then fixed with 4% PFA for 15 min.The immunofluorescent density was determined under the Zeiss confocal microscope.
The mitochondrial superoxide was detected by using MitoSOX Red (Invitrogen).Brain slices isolated acutely were incubated for 4 h in carboxygenated ACSF at 32 °C.5 μM MitoSOX Red was added to the brain slices and allowed for incubation for 10 min.Slices were then fixed with ice-cold 4% PFA in PBS overnight at 4 °C and cut into 30 μm sections.Brain slices were mounted onto slides with a mounting medium containing DAPI (Vector Laboratories).Slices were imaged using a Zeiss confocal microscope.All parameters (pinhole, gain, contrast, offset) were held constants for all sections from the same experiment.

Mitochondrial membrane potential (ΔΨm) assay
The ΔΨm was measured using the Mitochondrial Membrane Potential Assay Kit with TMRM (Beyotime, C2001S) following the manufacturer's instructions.The TMRE probe was diluted with neurobasal A culture medium and incubated with cultured cells for 30 min.Cells were imaged using a Zeiss fluorescence microscope.All parameters (pinhole, gain, contrast, offset) were held constant for all sections from the same experiment.